Apolipoproteins are the major protein component in soluble lipoprotein complexes with apolipoprotein A-I (Apo A-I) being the major protein component in high density lipoprotein (HDL) particles.
The apolipoproteins of the A, C and E families have evolved from a common ancestral gene and are structurally similar. These protein molecules generally contain a series of 22-amino acid tandem repeats that are often separated by proline residues. The repeating 22-amino acid segments form amphipathic α-helices which enable binding to both lipid and water surfaces. In the case of human Apo A-I (243 amino acids; 28.1 kDa) there are eight 22-mer and two 11-mer amphipathic helices (Lund-Katz & Phillips, 2010, Subcell Biochem. 51, 183-227). The amphipathic α-helices of the apolipoproteins play a critical role in stabilising the lipoprotein. They do this by orientating the apolipoprotein so that the predominantly hydrophobic helical faces can interact with the hydrophobic lipids in the complex whilst the opposing predominantly hydrophilic faces of the apolipoprotein interact with the surrounding aqueous environment. However when these proteins are separated from the lipid component, exposure of the hydrophobic amino acid residues to an aqueous environment can make them difficult to handle. In particular the hydrophobic faces of the α-helices have a tendency to self-associate resulting in aggregate formation and in some conditions precipitation. For example, a 1 mg/mL solution containing Apo A-I Milano is estimated to contain 80% of the protein in an aggregated form when stored in 50 mM phosphate buffer at pH 7.4 (Suurkuust & Hallen, 2002, Spectroscopy 16, 199-206).
Apo A-I is synthesized by the liver and intestine and is responsible for the physiological function of HDL in the blood; the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as “reverse cholesterol transport” (RCT). As a consequence the HDL particles are present in plasma in a range of sizes and are continually remodelling due to these RCT lipid transfer activities. Thus HDL particles are characterized by a high density (>1.063 g/ml) and sizes ranging from about 5 to 20 nm (Stoke's diameter). The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. Hence, Apo A-I in HDL is thought to have an anti-inflammatory function and to restrain the occurrence and development of CHD. Furthermore, Apo A-I has shown to decrease the Low Density Lipoproteins (LDL) level in the blood and is known to bind to lipopolysaccharides or endotoxins, thus having a major role in the anti-endotoxin function of HDL. The “protective” role of HDL and Apo A-I as the primary protein constituent has been confirmed in a number of studies. High plasma levels of Apo A-I are associated with a reduced risk of CHD and presence of coronary lesions. Apo A-I is thus promising for applications in drugs like reconstituted HDL for applications in acute coronary syndromes, atherosclerosis treatment, anti-inflammation treatment, antitoxin treatment, liver-targeting drugs, etc.
Biological therapeutics of either recombinant or plasma origin are commonly manufactured using biological feed-stocks that are intrinsically contaminated with pathogens such as viruses. Moreover, some manufacturing processes are, by their nature, susceptible to pathogen contamination from extrinsic sources. Accordingly, manufacturers of biological therapeutics are required to incorporate sufficient virus clearance steps into their manufacturing processes to ensure that their products are contaminant-free.
Biotechnology products (typically proteins or DNA) are produced with recombinant DNA in cell cultures, transgenic animals, or transgenic plants. Common cells used for production include Chinese Hamster Ovary (CHO) cells, E. coli bacteria, and yeast. Cell-based production systems are typically carried out in batch mode although a small number of perfusion systems are also in use. Final commercial scale fermentation is carried out at 1,000-100,000 L scale with the majority of CHO based fermenters in the 8,000-25,000 L scale.
Human blood plasma is nowadays collected in large amounts (for example, it has been estimated that in 2010 that 30 million liters of plasma were collected worldwide) and processed to individual fractions; some of these fractions contain the apolipoprotein, Apo A-I. Examples of such plasma fractions include Cohn Supernatant I, Cohn Fraction II+III, and Cohn Fraction IV (e.g. Cohn Fraction IV-1) or variations thereof (e.g. is a Kistler/Nitschmann Fraction IV). Since blood and plasma potentially contain transfusion-transmissible pathogens, such pathogens, in particular viruses must be removed or inactivated when blood- or plasma-derived components are used as therapeutics or as a vehicle for therapeutic delivery. However, viruses are often not easily removed and may still be present in plasma-derived components, even if they are highly purified. In particular, small non-enveloped viruses such as Picomaviridae (e.g. hepatitis A virus) which have a size of about 27-32 nm and Parvoviruses which have a size of about 18-26 nm, are of special concern. This is due to both their small size and their high physiochemical stability. Thus, there is an ongoing need for the development of methods that allow for efficient virus removal or inactivation of plasma-derived protein therapeutics.
Common virus inactivation technologies include physical methods such as the classical pasteurization procedure (60° C. heating for 10 hours), short wavelength ultra-violet light irradiation, or gamma irradiation and chemical methods such as solvent detergent or low pH incubation. Virus removal technologies include size exclusion methods such as virus filtration which is also often referred to as nanofiltration. These virus filtration methods have been shown to be effective methods for removing viruses from protein solutions.
Virus filtration has the benefit of being a mild method for removing viruses from protein solutions, and generally allows for a high level of protein recovery and the biological activity of the proteins to be fully preserved. Optimal virus filters must maximize capacity, throughput, and selectivity. The capacity of a virus filter is the total volume of filtrate per m2 of filter surface area that can be processed before the flux declines to an unacceptably low value during constant pressure filtration. Throughput refers to the speed at which the feed can be filtered (maximum sustainable permeate flux). Selectivity refers to the ability to yield high product recovery and high virus particle retention. These filters must be able to process the entire bulk feed at acceptable filtrate fluxes, reject virus particles, and maximize protein passage. Fouling during virus filtration is typically dominated by protein aggregates, DNA, partially denatured product, or other debris.
Filter manufacturers often assign terms like nominal or mean pore size ratings to commercial filters, which usually indicate meeting certain retention criteria for particles or microorganisms rather than the geometrical size of the actual pores.
For viral clearance, filtration is conducted through a filter membrane, which has a nominal pore size smaller than the effective diameter of the virus which is to be removed. The presence of only a small number of abnormally large pores (300 kDa or larger nominal molecular weight cutoff, NMWCO) will permit excessive virus leakage. Hence virus filters must be manufactured so as to eliminate all macro-defects. This is typically accomplished through the use of composite membranes that provide the required combination of virus retention and mechanical stability. Virus-removing filter membranes are typically made from materials such as regenerated cellulose, for example a cuprammonium-regenerated cellulose or synthetic polymer materials like hydrophilic polyvinylidene fluoride (PVDF) or hydrophilic polyether-sulfone (PES) as described in the literature: Manabe. S, Removal of virus through novel membrane filtration method, Dev. Biol. Stand, (1996) 88: 81-90; Brandwein H et al., Membrane filtration for virus removal, Dev Biol (Basel), (2000) 102: 157-63; Aranha-Creado et al., Clearance of murine leukaemia virus from monoclonal antibody solution by a hydrophilic PVDF microporous membrane filter, Biologicals. (1998) June; 26 (2): 167-72; Mocé-Llivina et al., Comparison of polyvinylidene fluoride and polyether sulfone membranes in filtering viral suspensions, Journal of Virological Methods, (2003) April, Vol. 109, Issue 1, Pages 99-101.
Virus filtration methods that have been described include WO96/00237 which relates to a method of virus-filtering a solution that contains macromolecules (i.e. protein) by adding salt to the solution to a level of at least 0.2 M. The applicants recommend using salts that exhibit the high salting-out effect that is characteristic of the high end of the Hofmeister series, in particular citrate, tartrate, sulfate, acetate or phosphate anions and sodium, potassium, ammonium or calcium cations. Sodium chloride is particularly preferred, and salts that exhibit a low salting-out effect at the low end of the series (e.g. GuHCl) are not used.
Consistent with WO96/00237, Kim et al (Biotechnology & Bioprocess Engineering, 2011, 16, 785-792) describe the virus filtration of Apo A-I in the presence of sodium chloride (250 mM NaCl, 30 mM Tris at pH 8). This step is carried out immediately after elution from a DEAE-FF column. The propensity for Apo A-I to aggregate however imposes a manufacturing limitation in that the filtration step ideally needs to be completed either as the Apo A-I is eluted from the column or alternatively the Apo A-I needs to be stored in the presence of salt at very low protein concentrations (e.g. 0.1 mg/mL). This later approach has the disadvantage of then requiring overly large filtration volumes. The cost of virus filters is substantial. Thus any reduction in filter capacity due to for example apolipoprotein aggregation or overly large filtration volumes can result in significantly higher processing costs at commercial scale.
WO03/105989 relates to the use of clathyrate modifiers such as polyol sugar or sugar alcohol (i.e. sucrose and sorbitol) and is aimed at increasing the hydrophobicity of the filter membrane surface and decreasing the hydrodynamic radius of the protein as well as reducing the tendency for the self-association of the protein desired to be filtered.
US 2003/232969 A1 relates to a method for removing viruses from solutions of high molecular weight proteins like fibrinogen (340 kDa) by nanofiltration.
Apolipoproteins like Apo A-I being relatively small (28 kDa) should be readily amenable to virus filtration. However as already described above their hydrophobic nature along with their unfortunate tendency to form aggregates promote the formation of protein clusters on the filter surface and also to clogging the pores of the filter. In terms of the filter itself, this can occur on the upper surface of the membrane, both by pore blockage and/or by the formation of a cake or deposit, and also within the membrane pore structure. Fouling causes decay in flow rate for constant pressure operation and increases the pressure for operation at constant filtrate flux. As a result of filter fouling there can be reductions to the selectivity of the filtration resulting in lower protein recoveries and/or lower virus retention. Additionally filter fouling reduces the capacity and throughput resulting in longer filtration times and/or the requirement for increased filter area. In addition, operating conditions which are optimal for maintaining apolipoprotein solubility and preventing aggregation might not be optimal for ensuring a high viral clearance. In particular chaotrophic substances that might be used to stabilise the apolipoprotein may alter the filterability properties of the pathogen (e.g. virus) and possibly also the filter membrane. As a consequence, the presence of a chaotrophic substance at particular concentrations might also allow unwanted virus penetration across the filter membrane, thus negating the usefulness of the step for processing solutions comprising apolipoproteins.
It is therefore an object of the present invention to provide a filtration method for safely removing viruses, in particular small non-enveloped viruses such as parvoviridae, which is suitable for solutions comprising apolipoproteins, like Apo A-I, and which is also suitable for industrial application.